Copyright 2001 by the Genetics Society of America

Dual-Tagging Gene Trap of Novel Genes in Drosophila melanogaster

Tamas Lukacsovich,*,† Zoltan Asztalos,†,1 Wakae Awano,† Kotaro Baba,‡,2 Shunzo Kondo,§

Suguri Niwa† and Daisuke Yamamoto*,†

*School of Human Sciences and Advanced Research Institute for Science and Engineering, Waseda University, Saitama359-1192, Japan, †ERATO Yamamoto Behavior Genes Project, Japan Science and Technology Corporation ( JST) at

Mitsubishi Kasei Institute of Life Sciences, Tokyo 194-8511, Japan, ‡Department of Physics, University of Tokyo,Tokyo 113-0033, Japan and §Mitsubishi Kasei Institute of Life Sciences, Tokyo 194-8511, Japan

Manuscript received August 18, 2000Accepted for publication November 3, 2000

ABSTRACTA gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the

gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion,i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporalexpression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-stepscreening process using two independent markers, mini-w and Gal4, each indicating the integration ofthe vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacksa polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of thevector integration site in the genome. However, the eyes of flies are not orange or red unless the vectoris incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene andpolyadenylated at the 39 end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only whenit is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readilycloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsiblefor mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloningof trapped genes.

THE enhancer-trap method is widely used in Dro- functional information on nearby genes, although theysophila for identifying new genes on the basis of are quite useful in monitoring the enhancer activity near

their expression patterns (O’Kane and Gehring 1987; the vector insertion site. In many cases, this expressionBellen et al. 1989; Bier et al. 1989; Wilson et al.1989). pattern resembles that of the transcript of an intrinsicIt is also used for generating cell- or tissue-specific mark- gene. The gene, however, may lie over tens of kilobasesers or for manipulating a particular cell type by targeted away from the insertion point. Under such circum-ectopic expression of transgenes (Brand and Perrimon stances, cloning and analyzing the gene under consider-1993; Bello et al. 1998). Most enhancer-trap insertions ation could be a time-consuming process. An even moreresult in some reporter gene expression because the complicated situation may arise in some enhancer-trapP-element-based vector constructs tend to become inte- lines in which the reporter gene expression is influ-grated into the 59 regulatory regions of functional tran- enced by multiple enhancer elements of different tran-scription units (Spradling et al. 1995) and thus the scription units (Bolwig et al. 1995).promoter of the inserted reporter is activated by a To circumvent these problems inherent in the en-nearby enhancer sequence. In spite of the high inci- hancer-trap system, we developed a gene-trap methoddence of reporter gene expression, many of these en- that utilizes a new vector construct. The primary re-hancer-trap lines do not exhibit detectable mutant phe- porter Gal4 gene in this vector lacks a promoter. There-notypes. That is, the genomic site of integration is under fore, this reporter gene is expressed only when its mRNAthe influence of a particular enhancer sequence, yet is covalently ligated to an endogenous mRNA either bywith little effect on transcription of the adjacent gene. splicing or by readthrough transcription. The patternThus, the enhancer-trap lines typically do not provide of reporter gene expression exactly matches that of the

trapped gene since its transcription starts from the pro-moter of this gene. All trap lines are mutants becauseCorresponding author: Daisuke Yamamoto, School of Human Sci-transcripts of the trapped gene are likely to lose theirences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama

359-1192, Japan. E-mail: daichan@mn.waseda.ac.jp function as a result of fusion with the inserted sequence.1 Present address: Department of Genetics, University of Cambridge, The gene-trap system presented here invariably yieldsDowning St., Cambridge CB2 3EH, United Kingdom.

fusion products of the reporter and an exon sequence2 Present address: National Institute of Sericultural and EntomologicalScience, Tsukuba, Ibaraki 305-8634, Japan. of the inserted gene. Such fusion products in gene-trap

Genetics 157: 727–742 (February 2001)

728 T. Lukacsovich et al.

hs-neor is inserted between the Gal4 and the mini-w genes. Tomutants provide us with a much easier and faster cloningensure translation of Gal4 for the forward reading frames, themethod than any of the currently available enhancer-stop-start signal (see Le et al. 1991) is placed immediately

trap methods. upstream of the Gal4-coding region. A splicing donor site,Few studies have been directed toward developing which connects mini-w to a downstream exon of the host gene,

is inserted in the 39 end of the mini-w coding region. TheDrosophila gene-trap systems (Dunn et al. 1993). Thisdetails of vector construction are available upon request. Theis presumably because of the expectation that gene-nucleotide sequence of the entire vector has been submittedtrap systems would provide much fewer positive linesto the DDBJ database under the accession no. AB028139. A

exhibiting reporter gene expression than enhancer-trap schematic map of the construct and the principle of its opera-systems. In the enhancer-trap system, the reporter gene tion in a gene-trap system is illustrated in Figure 1.

We also prepared a variant construct, pGTD-b. In this con-is expressed regardless of its orientation relative to thestruct, the full-length Gal4 sequence in pGT1 is replaced withinserted gene, whereas in the gene-trap system, the vec-the sequence of the Gal4-binding domain followed in frametor needs to be integrated in the same orientation asby the coding sequence of the tumor suppressor gene p53.

the trapped gene (see Figure 6A and the relevant de- The flies carrying pGTD-b are unable to express transgenesscription in the text for orientation sensitivity of the driven by UAS, unless they also carry the second construct,

pGTD-a, which contains the sequence encoding the activationgene-trap vectors). Therefore, unless a reliable detec-domain of Gal4 fused to the SV40 large T-antigen, a bindingtion method for gene-trap events is available, more stud-target of p53. Transcription of this fusion gene is controlledies need to be carried out to obtain a comparable num-by the hsp70 promoter. These constructs provide a means of

ber of positive lines. To overcome this problem, we temporal control of Gal4 activity in the gene-trap lines. Thisuse two independent markers, mini-white (w) and Gal4 is because the functional Gal4 protein is produced only when

p53 binds to the SV40 large T-antigen, which is inducible bygenes, as a “dual tag” for the trapped gene. In the firstheat shock.screening step, flies with intense eye color are chosen

Cloning of trapped genes: cDNAs for the trapped genes areas the most likely candidates for successful gene-trapobtained in three consecutive steps (Figure 2). The first step

events. These lines are then checked for in vivo Gal4 uses the vectorette PCR technique (Arnold and Hodgsonexpression by crossing them with a fly line harboring 1991; Allen et al. 1994) for a mRNA template. Poly(A)1RNAsa UAS-driven luciferase gene, the product of which is extracted from a gene-trap strain are converted into cDNAs

from which a chimeric cDNA composed of the Gal4 or mini-wdetectable with a luminometer (cf. Brandes et al. 1996).reporter and the exonic part of a host gene is amplified usingA double-marker system has been employed in theprimers corresponding to the adaptor sequence and the re-mouse gene-trap technique (Friedrich and Soriano porter sequence. The exonic sequence, thus obtained, is used

1991), in which the b-galactosidase and neomycin resis- in a database search for identical expressed sequence tagtance genes are typically coupled. A more recent version (EST) sequences.

When the search does not identify the cloned sequence inof a mouse gene trap uses a vector containing two re-the database, we perform rapid amplification of cDNA endsporter genes. One of these reporters has a promoter(RACE) with multiple cDNA libraries to obtain a full-lengthbut lacks a polyadenylation signal sequence, while the cDNA. The total recombinant l-phage DNAs prepared from

other has a polyadenylation signal sequence but lacks each of several cDNA libraries are used as templates for PCRa promoter (Zambrowicz et al. 1998). The promoterless analysis. In the first round, four parallel PCRs are run with

the primer corresponding to one strand of the expressedsequence is preceded by an artificial splice acceptor site,sequence, paired with either the forward primer or reversewhile the promoter-controlled sequence is followed byprimer with a lgt10 or lgt11 (depending on the actual library)an artificial splice donor site. These two sites are in-sequence. If the library contains a cDNA homologous to the

volved in the production of two chimeric mRNAs. This primer sequence, the cDNA appears in two pieces, one corre-gene-trap system for the mouse, designed for large-scale sponding to the 59 part of the cDNA and another to the 39mutagenesis, is thus constructed on the basis of a princi- part. These PCR products can be sequenced either directly

by using another nested primer or after insertion into a plas-ple similar to that of our system for Drosophila. Here,mid. By using this method, many different cDNA librarieswe report on the technical details of the dual-taggingcan be checked in a single experiment, and a “suitable one,”gene-trap method together with some analyses of gene- containing the desired cDNA, can be determined easily.

trap fly lines. In the third step, the final PCR is performed to obtain thefull-length product using the library chosen as the template.The primers used here correspond to the 59 and 39 end se-quences of the cDNA obtained as described above.MATERIALS AND METHODS

Mutagenesis and mutant screening: The jump-start method(Cooley et al. 1988) is used for mutagenesis with the gene-Construction of vectors for the gene-trap system: The pGT1

vector (Figure 1A) is constructed by assembling three markers trap (pGT1 or pGTD-b) vector as a mutator and P(ry1D2-3)transposon as a jump starter, i.e., the source of transposase(i.e., mini-w, Gal4, and hs-neor), splicing sites, and the “stop-

start” signal into the pCaSpeR3 vector (Pirrotta 1988). (Figure 3). All the flies used for mutagenesis have a w2 back-ground. With this background, the original pGT1 or pGTD-bpCaSpeR3 in its original form harbors an eye-color marker

mini-w, which is conserved in pGT1 except for its 39 untrans- insertion confers only a very faint eye color, presumably be-cause of the instability of the vector-derived w1 mRNA, whichlated region. A Gal4-containing DNA fragment is excised from

pGaTB (Brand and Perrimon 1993) and inserted into the lacks polyadenylation signals. When the vector is properlyintegrated into a gene, the mini-w gene in the vector is splicedmodified polylinker site of pCaSpeR3. This fragment also con-

tains the hsp70 transcriptional terminator. The third marker to an exon 39 to the integration site and is polyadenylated.

729Gene Trap in Drosophila

Figure 1.—Schematic representation of the principle of trapping genes by the dual-tagging gene-trap method. (A) A map ofthe pGT1 vector. The position and direction of Gal4, hs-neor, and mini-w genes are indicated by thick arrows. hspT representsthe hsp70 terminator. 39P and 59P are the P-element ends. The locations of the stop-start signal, splice donor site, and restrictionsites are illustrated. (B) The structure of the gene-trap vector pGT1. A solid box represents the hsp70 terminator; an open circle,the splice acceptor site; and a solid circle, the splice donor site. When integrated into a gene in the Drosophila genome (C),pGT1 operates to produce two fusion transcripts, one encoding the GAL4 sequence fused to the 59 portion of the inserted gene,and the other encoding mini-w fused to the 39 portion of the gene (D). The fusion mRNA is translated into the functionalproteins Gal4 and White (E), the former of which can activate transcription of a cDNA placed under the control of UAS (F),while the latter confers eye pigmentation in the fly signifying successful gene trapping.

730 T. Lukacsovich et al.

Figure 2.—Schematic rep-resentation of the steps of Vect-orette RT-PCR. The adaptor-ligated, double-strand fusioncDNA is subjected to tworounds of PCR (A), yieldingthe product illustrated in B.The synthetic adaptor is shownby a thick line (the length notin scale). The primers corre-sponding to partial sequencesof the host gene are used incombination with the vector-specific primers in two subse-quent rounds of PCR on thetemplate with the entire cDNAprepared from a whole library(C). The products of these re-actions provide the sequenceinformation for the search forcorresponding ESTs. The dot-ted portion represents the se-quence matched to an EST.(D) The two PCR products ob-tained from the previous step,representing the 59 and 39 endsof the trapped cDNA, respec-tively. After determination oftheir sequences, another PCRis performed to get the full-length cDNA. (E) The finalproduct.

Therefore, collection of flies with dark eye color after remobili- refer to each fly line using numbers placed after either GTor GTD, depending on the inserted vector in the fly’s genome.zation of pGT1 or pGTD-b allows recovery of chromosomes

with the vector inserted into genes. The numbers of jump-start crosses, established lines, and linespositive for Gal4 expression are summarized in Table 1.Female flies heterozygous for the insertion of pGT1 or pGTD-b

on the X or second chromosome are crossed with male flies After the establishment of fly lines with a new insertion,they are crossed with another line carrying the UAS-luciferasecarrying Sb, D2-3 on the third chromosome over the TM3, Ser

balancer. We use fly lines pTrap1-2(pGT1) or G4-DBD-p53#6- gene (cf. Brandes et al. 1996). Females carrying the UAS-luciferase reporter gene are mated with males of a putative gene-1(pGTD-b) with a second chromosome insertion and exp7-

65(pGT1) with an X chromosome insertion as mutators that trap strain and transferred to a vial with fresh food containing300 ml of 1 mm luciferin. The females are allowed to lay eggsyield P-insertion lines at rates of 20% (pTrap1-2), 15% (G4-

DBD-p53 6-1), and 8% (exp7-65) (Table 1). This cross yields for 2 hr. The luciferase activity in larvae from these eggs ismeasured using a Luminescencer JNR AB-2100 (Atto, Tokyo).F0 “jump-start” males carrying both the gene-trap vector and

the D2-3 element. In these males, the vector gets mobilized In the embryonic and pupal stages during which luciferaseassay is impossible, a fly line carrying the UAS-GFP reporterand “jumps” into new insertion sites. When the vector is inte-

grated into a transcription unit, the F0 males should have dark gene is used for detecting Gal4 expression by green fluorescentprotein fluorescence (Chalfie et al. 1994; Yeh et al. 1995).mosaic eyes. Each of these males is chosen as the founder of

a strain and is crossed with w2 females having both the CyO Scanning electron microscopy (SEM): The flies are preparedfor critical point drying and coated with a 2-nm layer of gold.and TM3 balancers. When necessary, the FM7c balancer is

used to maintain the X chromosome insertion. The balancers Images are obtained using a low-voltage prototype SEM asdescribed by Miyamoto et al. (1995).used are those described by Lindsley and Zimm (1992). We

731Gene Trap in Drosophila

Figure 3.—Mating scheme for generating gene-trap fly lines. Mutator females carrying the gene-trap vector (pTrap) insertedinto the second chromosome are mated with the males carrying the transposase source D2-3 in the third chromosome. All fliesused are white-eyed (w). Each F1 male with mosaic eyes is mated with five females, the third chromosome of which is balancedby Sb/TM3 Ser. F2 flies with pigmented eyes bear the pTrap insertion in either of the chromosomes indicated by asterisks. Theinserted chromosome can be identified in F3. The flies with pigmented eyes that do not carry Cy are discarded to exclude thepossibility of recovering the chromosome with the original insertion. Each F2 male or female with pigmented eyes is mated withfemales or males having the balanced third chromosome (Sb/TM3 Ser). Offspring of this cross carry the mutated chromosomederived from one parent. When eye pigmentation of F3 is always associated with CyO, the insertion has taken place in the CyObalancer. Otherwise, the insertion is present in the X or third chromosome. (We exclude the short fourth chromosome in ourobservation.) When the F3 flies with Sb/TM3 Ser have pigmented eyes, the gene-trap vector must be inserted in the X chromosome.When they have white eyes, the insertion must be in the third chromosome. The gene-trap fly lines are established from the F3

flies using appropriate balancer chromosomes. The same scheme is used for generating mutant fly lines with the gene-trap vectorinserted in the X chromosome.

X-gal and immunochemical staining of eye discs: The tissue- sequence is also used as a probe to confirm the obtained mapposition, because a paralogous gene has been mapped tospecific expression pattern of the trapped gene is determineda different site. Signal detection is performed on polyteneafter introducing a UAS-lacZ construct (instead of UAS-lucifer-chromosomes using an anti-DIG antibody as described pre-ase) into the gene-trap fly lines. Staining with X-gal is per-viously ( Juni et al. 1996).formed as described previously (O’Kane 1998). Staining with

an anti-b-galactosidase antibody is carried out as described byMatsuo et al. (1997).

RESULTSIn situ hybridization to polytene chromosomes: The Gal4-coding sequence of the gene-trap vector is labeled with digoxi-

Structure and principle of operation of the gene-trapgenin (DIG)-11dUTP (Boehringer Mannheim, Indianapolis)vector pGT1: The structure of the gene-trap vectorand used as a probe to map the insertion site in the chromo-

some. In the case of the a-mannosidase II gene, the cDNA pGT1, is illustrated in Figure 1A. pGT1 is a P-element

732 T. Lukacsovich et al.

TABLE 1

The number of integration events of the gene-trap vectors into chromosomescompared among three different mutators

Mutator strains

Exp 7-65 pTrap1-2 G4-BDB-p53#6-1(pGT1 on X) (pGT1 on II) (pGTD-6 on II) Total

Mosaic-eyed males 675 460 420 1555used for generation offounders of strains

Established lines with X — 29 13 42an insertion in the X, II 23 21a 26a 70second (II), or third III 31 42 25 98(III) chromosome total 54 (8%)b 92 (20%)b 64 (15%)b 210 (14%)b

Lines with Gal4 activity 18 (33%)c 32 (35%)c 24 (38%)c 74 (35%)c

a The gene-trap vector was integrated into the CyO chromosome.b The ratio of the number of established lines over that of total jump-started mosaic-eyed males shown in

the top row.c The percentage of gene-trap lines with Gal4 expression relative to the total stocks with vector insertions,

which are given in the middle row.

vector containing three markers: Gal4, mini-w, and hs- way is tagged dually by Gal4 and mini-w, we call thismethod the “dual-tagging gene trap.”neor. The Gal4 gene lacks a promoter but has a poly(A)1

signal sequence. The mini-w gene has a promoter but The vector must be inserted into the genome beforeany attempt at its intragenic integration. The vectorlacks the poly(A)1 signal sequence. The Gal4 gene is

preceded by an artificial splice acceptor site, which contains hs-neor, which allows us to recover the flies withthe vector, even when the integration site is outsideallows the Gal4 sequence to be transcribed as a fusion

mRNA with an upstream exon sequence (Figure 1, B the transcription unit. In fact, the flies with the vectorinserted outside the transcription unit had faintly pig-and C). That is, Gal4 would never be expressed unless

the vector is integrated downstream of the promoter of mented eyes (pale yellow), which made it possible torecover these flies without using hs-neor (Figure 4D).a host gene. Since the stop-start sequence is placed

between the splice acceptor site and the Gal4 gene, the Gene-trap screens: The primary advantage of thedual-tagging gene-trap system is its ability to distinguishopen reading frame (ORF) for Gal4 is maintained in

any integration event (Figure 1D). It is unequivocal that the P-element insertions occurring downstream of thepromoters (i.e., intragenic insertions) from those oc-the Gal4 fusion protein activates transcription from the

target sequence UAS (Figure 1E). On the other hand, curring in other regions of the genome simply by evalu-ating the eye color intensity of the flies. For proceduralthe mini-w gene is followed by an artificial splice donor

site, which is involved in the production of a chimeric convenience, the degree of eye pigmentation is gradedinto four levels, 1 to 4, by comparing the eye colormRNA composed of the mini-w sequence and the exonic

sequence of a host gene 39 to the vector integration site. of individual flies with that of four arbitrarily chosenstandard strains; the eye color of each strain representsThis chimeric mRNA is polyadenylated and encodes the

functional White protein (Figure 1C). Even when the one of the four pigmentation levels (Figure 4D). Inthe secondary screening of the flies in which a gene isvector is inserted outside a gene, the mini-w gene can

be transcribed from its own promoter. In this case, how- trapped, we examine the expression of another marker,Gal4. Gal4 activity is detected on the basis of UAS-lucifer-ever, the mini-w mRNA would not be polyadenylated,

because its 39 end cannot be spliced to an exon of the ase expression. The females of each strain are allowedto lay eggs on luciferin-containing food, and the off-host gene. Such an unpolyadenylated mRNA is likely to

be degraded rapidly before being translated, thereby spring are subjected to luminescence measurements atthe first, second, and third instar larval stages. Live lar-resulting in the absence of eye pigmentation in the fly

carrying the vector (the flies have a w2 background). vae are subjected to the measurements. Luminescenceintensity higher than the background level is detectedIn contrast, the mini-w gene is expected to confer a dark

eye color on the fly when the vector is integrated into in some of the strains (Figure 4A). To ascertain that thelines with luciferase activities express Gal4, the putativea gene, because the chimeric mRNA is polyadenylated.

Thus, the two reporter genes, Gal4 and mini-w, generate gene-trap lines are crossed with flies, carrying UAS-lacZ,by which histochemistry of b-galactosidase activity canfunctional proteins only when the vector is integrated

into a transcription unit. Since the gene trapped in this be used to monitor Gal4 expression. In no case have

733Gene Trap in Drosophila

Figure 4.—Correlationbetween the degree of eyepigmentation and the levelof Gal4 expression in Dro-sophila strains with a gene-trap-vector insertion. (A)Relationships between thedegree of eye pigmenta-tion and the level of lumines-cence that indicates Gal4 ex-pression. Ninety-nine strainswith vector insertions areclassified into either threegroups on the basis of thelevel of luminescence (L, rel-ative counts for 20s deter-mined using the lumino-meter; 0 , L , 100, 100 ,L , 1000, and 1000 , L)or into four groups on thebasis of the eye color gradeas illustrated in D. Lumines-cence reflects luciferase ac-tivity as induced by the UAS-luciferase reporter gene,which is activated by Gal4derived from the gene-trapvector inserted into thegene. Results are obtainedat the third instar larvalstage. The degree of eyepigmentation is indicatedby the color (examples areshown in D). In the left partof the graph in A, the num-ber of fly strains that belongto either one of four eyecolor groups (illustrated bya color code) is shown forthree luminescent groups.In the right part of thegraph in A, the relative con-tribution of strains with dif-ferent luminescence levelsin each eye color group isdepicted. (B) The percent-age of strains with detect-able levels of Gal4 expres-sion is shown for fourgroups of strains on the ba-sis of the degree of eye pig-mentation, i.e., grade 1 tograde 4. The strains exhib-iting 100 or higher lumines-cence counts are consid-ered to be positive for Gal4

expression. Approximately90% of the strains belonging to the grade 4 group with the darkest eye color show Gal4 expression.(C) The percentage of fly lines with different degrees of eye pigmentation. Ten percent of flies generated from mutageniccrosses belong to grade 4 group. (D) The eyes of “representative” strains of the four eye color grade groups.

luciferase and b-galactosidase activities dissociated; thus, be correlated with intense pigmentation of the eye.Note, however, that the expression patterns of Gal4 andthe luciferase reporter is proven to be a reliable and effi-

cient tool for mass screening of Gal4-expressing strains. mini-w are usually different, because the Gal4 expressionis driven by the intrinsic promoter of the trapped geneWhen the trapped gene is tagged dually by mini-w and

Gal4, the presence of Gal4 expression is considered to whereas mini-w expression is controlled by its own pro-

734 T. Lukacsovich et al.

moter. Thus, the mini-w gene is expressed only in the (SD) sites work as expected, and the endogenous mRNAis found to be fused to the Gal4 (and mini-w) sequenceseye, but Gal4 is expressed wherever the trapped gene

is expressed. exactly at the expected nucleotide (Figure 5). Figure 5also includes examples where upstream, secondaryMore than 200 strains with gene-trap-vector insertions

are generated from 1550 males having the mosaic eye splice sites are used (GTexp16-#8 and GTDexp1-#2/2,Figure 5D). At nucleotides 15 and 24 upstream of thecolor due to P element mobilization (Table 1). Both the

degree of eye pigmentation and the level of luciferase designed splice acceptor site, there are two additionalAG dinucleotides (SAs marked with an arrowhead inactivity are determined in about half of the strains to

see whether the expected correlation exists. The fly Figure 5A) that are 100% conserved in the 39 end ofDrosophila introns (Mount et al. 1992). This part comesstrains with gene-trap vectors are classified into three

classes on the basis of their levels of luminescence, L from the 39-end sequence of the P element. In somegene-trap lines, the splicing occurs at either of these sites(Figure 4A), and the proportion of strains with four

different eye color grades is assessed in each of the (Figure 5D, arrowheads). In a few cases, two differentsplicing products are obtained from the same line (Fig-classes. The actual number of strains that were grouped

according to L levels and degree of eye pigmentation ure 5D, lower two rows).In another line, GTexp7-#45, the flanking sequenceis shown in the left half of Figure 4A. The percentage

of strains with different L levels within the same eye of the P-element insertion point is found to be identicalto the Berkeley Drosophila Genome Project (BDGP) ESTcolor groups is compared in the histogram shown in

the right half of Figure 4A. The percentage of flies GM04742. Notably, however, the EST sequence corre-sponds to the first intron of the trapped gene, and notthat express Gal4 is compared among the four groups

classified according to the degree of eye pigmentation to an exon. The intron in question has been obtainedby genomic PCR, after recovering the fusion cDNA con-in Figure 4B. It is clear that the fly strains with darker

eye color are more likely to express Gal4 than those taining an exon of the trapped gene. Thus, the intronof the trapped gene is found to contain an independentwith lighter eye color: 88% of the strains in group 4

express Gal4, whereas 48% of group 1 do (Figure 4B). gene on the complementary strand identical to GM04742. The gene-trap construct is inserted into the firstThe proportion of the strains with grade 4 eye color is

z10% of the total P-element-insertion stock generated untranslated exon of the gene registered as GM04742,but in the opposite transcriptional orientation. This in-by this method (Figure 4C). Each line carries a single

gene-trap vector (Table 2), except for GTDexp1-#31/ dicates that the direction of transcription of the Gal4reporter gene of the construct is also opposite to that1-GTDexp1-#2/2, in which the D2-3 element has been

reintroduced intentionally to initiate a local jump of of GM04742 (Figure 6A). Thus, this line could be usedto indicate the positional specificity of the reporter genethe inserted pGTD-b. It is noted, however, that there are

fly lines carrying a gene-trap-vector insertion without expression in the gene-trap construct compared withthat in the enhancer-trap construct. If an enhancer-trapGal4 expression yet with a dark eye color (Figure 4A).

This suggests that a dark coloration of the compound vector is integrated into the same position, its reportergene expression would reflect the expression patterneyes is a reliable indication that a host gene has been

trapped even when no Gal4 expression is detected. of the gene GM04742 (or maybe both of the genes). Onthe contrary, in the gene-trap system, Gal4 expressionThus, the dual-tagging gene trap provides a simple and

efficient approach for the generation of mutants. follows the pattern of a gene more distantly located buton the same DNA strand, i.e., the trapped gene, the firstSixty-two Gal4-expressing lines are chosen from the

99 lines shown in Figure 4A for further analysis. Among exon of which is spliced to the Gal4 mRNA.The most complicated configuration of trappingthe 62 lines, homozygosity is found to be lethal in 18,

semilethal in 7, and associated with sterility in 3. Thus, events is found in the line GTDexp1-#31/1-GTDexp1-#2/2. In this case, the inserted element is duplicatedroughly half of the generated gene-trap lines exhibit a

lethal or sterile phenotype. The flies used in the cross during local hop with the aid of the D2-3 jump-starterchromosome. As a result, two P elements are found inhave the isogenized Canton-S genetic background that

is free of lethal mutations (provided by M. Yoshihara). the same intron of the same gene (Figure 6C). Eachof the elements traps an exon upstream of it, leadingIn 4 other lines, the P element is inserted into the

CyO balancer; therefore, the effect of the insertions on to the identification of two transcription units. One ofthese two units is contained in the intron of the other.viability and fertility cannot be evaluated. The re-

maining 30 lines are maintained as homozygous stocks The former gene encodes a putative transcription factorwith a ring-finger motif, termed Brain tumor (Brat;without any difficulty.

Identification and characterization of trapped genes: EMBL/GenBank database accession nos. AF119332 andAF195870–73).Genes trapped by intronic integration of the vector: We have

cloned the partial sequences of 27 trapped genes listed Genes trapped by exonic integration of the vector: Thereare examples where the vector is integrated into thein Table 2. In most cases, the artificial splicing acceptor

(SA, marked with open arrows in Figure 5) and donor first untranslated exon of a gene. It is obvious that the

735Gene Trap in Drosophila

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737Gene Trap in Drosophila

Figure 5.—Sequences aroundthe fusion points of chimericcDNAs obtained in some gene-trap lines. (A) Shown are thesequences of the trap vectorsurrounding the splice ac-ceptor sites (SA, arrowheadsand an open arrow; upper row)or the splice donor site (SD;lower row) following the splic-ing signal sequence (boxed).The first two SAs (those indi-cated by arrowheads) are notintroduced intentionally intothe construct. These two sitesare originally contained in theP-element sequence and inci-dentally recognized as SAs bythe splicing machinery. Thestop-start sequence is under-lined. The region indicated bylowercase letters represents theP-element sequence. The vec-tor sequences are shaded. (Band C) The fusion junctions ofthe chimeric mRNAs from twoknown loci, anterior open (aop;B) and trithorax (trx; C). Thetop row shows the sequence ofwild-type cDNA at the junctionof the first and second intron.The junction is indicated by anarrow. The second row illus-trates the junction of the firstexon of the aop or trx gene andthe vector sequence adjacentto the stop-start and Gal4 se-quences. The third row showsthe junction of the first exonof the aop or trx gene and themini-w sequence. The se-quence of stop-start Gal4 ormini-w is shaded. (D) Examplesin which either of the first twoSAs is responsible for splicingthat leads to the fusion events.In the GTexp16-#8 line, thegene encoding phosphofructo-kinase is spliced to the vectorsequence at the second SA (ar-rowhead). In the trap event

GTDexp1-#2/2 in the GTDexp-1-#31/1-GTDexp1-#2/2 line, an exon of the host gene is spliced to the vector sequence at thefirst SA (arrowhead) in addition to that at the third (open arrow) SA. (E) The GTexp7-#49 line provides an example ofreadthrough transcription. In this case, the first exon of the host gene is followed by the P-element sequence. The junction ofthe host and P-element sequences is indicated with an asterisk.

artificial splice acceptor site is not used in these cases Drosophila phosphofructokinase gene (AB023510–11),which harbors the vector in an untranslated exon (Fig-because of the absence of upstream exons. Instead, the

Gal4 gene is expressed by readthrough transcription ure 6B). In this line, another Gal4 fusion mRNA is alsodetected. This fusion mRNA is a result of the splicingfrom the nearby promoter. An example of this is found

in GTexp7-#49, in which the Drosophila homologue of event at the second SA. A distant upstream exon of thephosphofructokinase gene (Figures 5D and 6B) is identi-the mouse Fas-associated factor (FAF1, Chu et al. 1995)

is targeted (Figure 5E; AB013610). fied by this fusion mRNA. The phosphofructokinase geneeven has a third untranslated exon (Figure 6B).Another line, GTexp16-#8, shows readthrough tran-

scription of Gal4 from a nearby promoter of the putative Expression and phenotypic analysis: The gene-trap lines

738 T. Lukacsovich et al.

Figure 6.—Complex genomic arrangements observed in three gene-trap lines. (A) Gene-trap line GT exp7-#45. The P elementis integrated into an intron of a novel gene, which contains a second gene with a sequence identical to GM04742 in thecomplementary strand. (B) Gene-trap line GTexp16-#8. The P element impinges upon the second noncoding exon of the geneencoding phosphofructokinase. (C) Gene-trap line GTDexp1-#31/1-GTDexp1-#2/2. This line contains pGTD-b, which producesa Gal4-binding domain followed by p53 (Gal4-p53) instead of full-length Gal4 (see materials and methods). As a result of thelocal hop of the P element induced by the D2-3 chromosome, two insertions occur in tandem in the same intron of the

739Gene Trap in Drosophila

Figure 7.—Monitoring ofthe expression of trapped genesby the Gal4 reporter gene. Gal4reporter gene expression as de-tected by UAS-lacZ is shown inthree gene-trap lines, GTexp16-#8 (A–C), GTexp7-#77 (D–F),and GTexp49-#2 (G–I), whichrevealed expression in definedneural structures with uniquegeometry. The lacZ expressionis detected by anti-b-galacto-sidase antibody immunocyto-chemistry except for A, whichrepresents X-gal staining. A, D,and G represent dorsal views ofthe brain-ventral ganglia com-plex of the third instar larvae.B, E, and H are frontal viewsand C, F, and I are dorsal viewsof the adult brain. In GT-exp16-#8, Gal4 is preferentiallyexpressed in the mushroombody and the antennal lobe. InGTexp7-#77, Gal4 is expressedin different classes of neurons,including the giant visual in-terneurons (arrow) that ex-tend arborizations horizontally

(Strausfeld 1976). In the GTexp49-#2 line, prominent expression is observed in the antennal lobe in both larval (G) and adult(H and I) brains. In the larval ventral ganglia, longitudinal axon bundles and segmental motor neurons are clearly stained. MB,mushroom body; a, a-lobe; b, b-lobe; g, g-lobe; LPR, lateral protocerebrum; CX, calyx; GC, giant commisure; AL, antennal lobe.

are crossed with a strain carrying UAS-lacZ, which ex- and Rubin 1995; Rogge et al. 1995). The expression ofAop in developing eye discs has been documented inpresses b-galactosidase detectable either using the chro-

mogenic substrate X-gal or by immunohistochemistry. detail (Lai and Rubin 1992; Rebay and Rubin 1995;Yamamoto 1996): In the third instar eye disc, almostThe patterns of reporter gene expression are unique

in each gene-trap line but are similar among the speci- all undifferentiated cells located basally expressed theAop protein, whereas none of the differentiated conemens in the line. In cells exhibiting reporter gene ex-

pression, uniform cytoplasmic staining is observed, mak- cells and photoreceptors expressed this protein. Ourresults of staining experiments on eye discs using aning it possible to visualize even cellular structures with

complex geometry, such as axons and dendrites of neu- anti-Aop antibody coincide with those of previous obser-vations (Figure 8E). The reporter gene expression inrons, in addition to cell bodies (Figure 7). Dynamic

changes in the expression of trapped genes during de- the aopGT1 gene-trap line is found in these undifferenti-ated cells (Figure 8D). The eye discs of aopGT1 heterozy-velopment are monitored clearly (Figure 7).

The expression pattern detected by the Gal4 reporter gotes are subjected to double staining with an anti-b-galactosidase antibody for the detection of the reportergene matches the localization of the protein encoded

by the trapped gene. This is demonstrated for aopGT1 by expression and with mAb22C, a marker for differenti-ated neurons (Fujita et al. 1982). It is found that eachcomparing the protein localization, as determined using

a specific antibody, and Gal4 reporter expression. The antibody labels a distinct group of cells (Figure 8F).These results indicate that the Gal4 reporter in the gene-aop gene encodes an E-26-specific-domain transcription-

al repressor (Lai and Rubin 1992; Tei et al. 1992) that trap vector labels the cells in which the trapped geneis normally expressed.prevents cells from differentiating prematurely (Rebay

novel gene. The second insertion traps an exon of a different gene contained in an intron of the former gene. The insertionsites of the gene-trap vectors (triangle) and the exon-intron organization of the first trapped gene are schematized in 1. Shownin 2 are the spliced mRNA structures in the gene-trap lines (left) in comparison with those in the wild type (right). The dottedboxes indicate noncoding exonic sequences; the striped boxes show artificial splice sites. In each case, the direction of transcriptionis indicated by arrows. pr, promoter; E1, E2, E3, and E4, exons; IE1, an exon of the intraintronic gene; as, artificial splicingacceptor site; ds, artificial splicing donor site; 59P, 39P, P-element end sequences; EST, sequences that match with those in theBDGP EST library; env, pol, and gag are the genes in an inserted retroviral sequence; ATG, translation start codon.

740 T. Lukacsovich et al.

Figure 8.—The mutant phe-notype associated with a gene-trap allele of aop, aopGT1. (A–C)Scanning electron microscopicobservations of the compoundeyes of Canton-S wild type (A),aoppok3r5 (B), and aopGT1/aoppok3r5

(C). (D–F) The expression pat-tern of aop in a third instar eyedisc of a aopGT1/1 larva deter-mined by X-gal staining withthe Gal4 reporter (D and F) orby an anti-Aop antibody (E).(F) An eye disc doubly stainedfor b-galactosidase reporter ac-tivity with X-gal and for a neu-ronal antigen recognized bymAb22C10. The anti-Aop anti-body and X-gal staining labelsbasally located undifferenti-ated cells, whereas mAb22C10marks apical projections of dif-ferentiated photoreceptors.Anterior to the right.

The null mutations in the aop locus are embryonic phenotypically silent when mutated. Second, the expres-lethal (Nusslein-Volhard et al. 1984), as is the aopGT1 sion pattern of the reporter gene in gene-trap lines isallele. The hypomorphic aop mutants exhibit the rough- the same as that of the trapped gene. This is in contrasteye phenotype in varying degrees (Yamamoto 1996), to the case in the enhancer-trap system where the re-and aoppok1 has a strong eye phenotype among such via- porter gene expression merely reflects the activity ofble alleles (Tei et al. 1992). No aopGT1/aoppok1 adults are the nearest enhancer. Third, the gene-trap methodobtained, indicating that this allelic combination is le- allows us to identify unequivocally the affected genethal. When placed over a chromosome carrying a weak and to readily clone it, because the transcripts (as withallele, aoppok3r5, the aopGT1 mutation yields a few escaper cDNAs) have reporter tags that provide specific primeradults having the rough-eye phenotype (Figure 8B). The sequences for PCR.roughness of the eyes of aoppok3r5/aopGT1 adults is more While the gene-trap system has the above-mentionedsevere than that of any of the known aop mutants that advantages, all the important features of the conven-develop into adults (Figure 8C). These results suggest tional enhancer-trap system are also preserved. Due tothat aopGT1 represents a very strong, likely null, mutant the presence of the Gal4-coding sequence, any gene ofin the aop locus. interest can be expressed when introduced into the

gene-trap line in the form of a UAS fusion (Brand andPerrimon 1993). The gene-trap insert can be remobil-

DISCUSSION ized by crossing the D2-3 chromosome into the gene-trap lines to generate revertants by precise excision orThe advantages of the newly developed gene-trap sys-new alleles by imprecise excision (Cooley et al. 1988).tem over the conventional enhancer-trap system are

The crucial step in efficient mutagenesis using the gene-threefold. First, all Gal4 gene-trap lines obtained are, intrap vector is the collection of founder males from whichprinciple, mutants, because the trapped gene producesthe putative gene-trap strains are established by subse-aberrant chimeric transcripts. This is in contrast to thequent genetic crosses. The collection of the founder malesGal4 enhancer trap in which only z10% of the linesrelies primarily upon the dark eye pigmentation, fol-have some discernible phenotype. In the case of en-lowed by luciferase assays for the detection of Gal4 ex-hancer-trap lines, phenotypical silence may result simplypression. Of 27 strains analyzed molecularly, all have afrom insufficient disruption of the gene by the insert.gene interrupted by the gene-trap vector, which resultsWith the gene-trap construct, the functions of the

trapped gene are vitally impaired, even if the gene is in the production of a fusion mRNA composed of the

741Gene Trap in Drosophila

a tetracycline-dependent transactivator system. Development 125:host gene and Gal4. We did not study in detail the fly2193–2202.

strains with dark eye color but without detectable Gal4 Bier, E., H. Vassin, S. Shepherd, K. Lee, K. McCall et al., 1989Searching for pattern and mutation in the Drosophila genomeexpression. On the basis of the results of PCR, in twowith a P-lacZ vector. Genes Dev. 3: 1273–1287.of such strains pGT1 is found to be inserted into the 59

Bolwig, G. M., M. Del Vecchio, G. Hannon and T. Tully, 1995untranslated region of the gene without producing a Molecular cloning of linotte in Drosophila: a novel gene that func-

tions in adults during associative learning. Neuron 15: 829–842.chimeric mRNA of the host sequence and Gal4 (M.Brand, A. H., and N. Perrimon, 1993 Targeted gene expression as aUmeda, personal communication). However, the mini-

means of altering cell fates and generating dominant phenotypes.w gene in pGT1 is transcribed even in these fly lines. It Development 118: 401–415.

Brandes, C., J. D. Plautz, R. Stanewsky, C. F. Jamison, M. Straumeis likely that the mini-w gene is spliced to the downstreamet al., 1996 Novel features of Drosophila period transcription re-exon of the trapped gene and is polyadenylated, therebyvealed by real-time luciferase reporting. Neuron 16: 687–692.

resulting in the dark eye color. Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward and D. C. Prasher,1994 Green fluorescent protein as a marker for gene expres-The success of the new gene-trap system dependssion. Science 263: 802–805.upon several technical factors. The use of artificial splice

Chu, K., X. Niu and L. T. Williams, 1995 A Fas-associated proteinsites provides a means to generate two fusion mRNAs, factor, FAF1, potentiates Fas-mediated apoptosis. Proc. Natl.

Acad. Sci. USA 92: 11894–11898.mini-w and Gal4, each of which can function as an inde-Cooley, L., R. Kelley and A. C. Spradling, 1988 Insertional muta-pendent marker for integrating a transgene into a tran-

genesis of the Drosophila genome with single P-elements. Sciencescription unit (i.e., gene-trap event). Another technical 239: 1121–1128.

Dunn, J. M., K. E. Zinsmaier, B. A. Mozer and S. Benzer, 1993 Afactor that is crucial for the success of the present systemnew ‘gene trap’ system for Drosophila. Ann. Rep. Biol. Calif. Inst.is the development of the new RACE technology. TheTechnol. 1993: 206.

triad of vectorette RT-PCR, RACE using multiple cDNA Friedrich, G., and P. Soriano, 1991 Promoter traps in embryonicstem cells: a genetic screen to identify and mutate developmentallibraries, and PCR using a whole library proves to be agenes in mice. Genes Dev. 5: 1513–1523.collection of extremely powerful methods for cloning

Fujita, S. C., S. L. Zipursky, S. Benzer, A. Ferrus and S. L. Shot-the exon of the trapped gene that could be at a location well, 1982 Monoclonal antibodies against the Drosophila ner-

vous system. Proc. Natl. Acad. Sci. USA 79: 7929–7933.physically quite remote from the vector insertion pointJuni, N., T. Awasaki, K. Yoshida and S. H. Hori, 1996 The Om(1E)on the chromosome. ESTs instead of sequence-tagged mutation in Drosophila ananassae causes compound eye over-

sites can be determined in the screening when the gene- growth due to Tom retrotransposon-driven overexpression of anovel gene. Genetics 143: 1257–1270.trap system is used. Because the entire Drosophila ge-

Lai, Z.-C., and G. M. Rubin, 1992 Negative control of photoreceptornome has been sequenced and made available, coding development in Drosophila by the product of the yan gene, ansequences of a trapped gene can be identified easily ETS domain protein. Cell 70: 609–620.

Le, S. Y., B. A. Shapiro, J. H. Chen, R. Nussinov and J. V. Maizel,using some ORF-detecting software, such as Genie.1991 RNA pseudoknots downstream of the frameshift sites ofScreening for genomic sequences that produce domi- retroviruses. Genet. Anal. Tech. Appl. 8: 91–205.

nant phenotypes when misexpressed has been em- Lindsley, D. L., and G. G. Zimm, 1992 The Genome of Drosophilamelanogaster. Academic Press, San Diego.ployed in several laboratories (Rørth 1996; Rørth et

Matsuo, T., K. Takahashi, S. Kondo, K. Kaibuchi and D. Yama-al. 1998; Toba et al. 1999). The misexpression strategy moto, 1997 Regulation of cone cell formation by Canoe and Rascould be useful for deducing the potential roles of the in the developing Drosophila eye. Development 124: 2671–2680.

Miyamoto, H., I. Nihonmatsu, S. Kondo, R. Ueda, S. Togashi etoverexpressed sequence, while the gene-trap systemal., 1995 canoe encodes a novel protein containing a GLGF/could be used to define the role of the trapped gene on DHR motif and functions with Notch and scabrous in common

the basis of its loss-of-function phenotype, as in classical developmental pathways in Drosophila. Genes Dev. 9: 612–625.Mount, S. M., C. Burks, G. Hertz, G. D. Stormo, O. White etgenetics. Thus, these two systems are complementary to

al., 1992 Splicing signals in Drosophila: intron size, informationeach other for functional analysis of the genome. content, and consensus sequences. Nucleic Acids Res. 20: 4255–4262.We thank M. Yoshihara, G. M. Rubin, S. Kondo, and Y. Kai for

Nusslein-Volhard, C., E. Wieschaus and H. Kluding, 1984 Muta-materials and assistance. This study was supported in part by Specialtions affecting the pattern of the larval cuticle in Drosophila melano-

Coordination Funds for Promoting Science and Technology from the gaster. I. Zygotic loci on the second chromosome. Roux’s Arch.Science and Technology Agency of Japan and by Waseda University Dev. Biol. 193: 267–282.grant No. 200B-029 to D.Y. O’Kane, C. J., 1998 Enhancer-traps, pp. 131–178 in Drosophila: A

practical approach, Ed. 2, edited by D. B. Roberts. IRL Press,Oxford.

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